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Studies of P-glycoprotein Drug Interactions - Administrative Supplement for Undergraduate Summer Research

P-糖蛋白药物相互作用的研究 - 本科生暑期研究行政补充

基本信息

批准号：
10810072
负责人：
INA L URBATSCH
金额：
$ 0.75万
依托单位：
TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-01 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10810072
关键词：
ABCB1 gene ATP Hydrolysis ATP-Binding Cassette Transporters Acceleration Administrative Supplement Affinity Amber Amino Acids Antineoplastic Agents Area Aromatic Amino Acids Binding Binding Sites Biochemical Biological Biological Assay Biological Availability Biomedical Engineering Blood - brain barrier anatomy Cardiovascular Agents Cardiovascular system Cell membrane Cells Chemicals Clinical Codon Nucleotides Cryoelectron Microscopy Cytoplasm Cytoplasmic Tail Development Dimerization Disease Documentation Drug Binding Site Drug Combinations Drug Design Drug Efflux Drug Interactions Drug Kinetics Drug Metabolic Detoxication Drug Monitoring Drug Transport Drug resistance Drug usage Energy Transfer Engineering Environment Excretory function Fluorescence Fluorescence Spectroscopy Fluorescent Probes Funding Goals HIV/AIDS Hydrophobicity Intestines Kidney Kinetics Knowledge Laboratories Learning Ligand Binding Lipid Bilayers Liver Maps Measures Membrane Mental disorders Molecular Molecular Conformation Monitor Multi-Drug Resistance Nifedipine Nucleotides Paclitaxel Pharmaceutical Preparations Positioning Attribute Prazosin Property Pump Recombinant Proteins Rhodamine 123 Roentgen Rays Scientist Screening procedure Site Structure Surface System Technology Terminator Codon Tertiary Protein Structure Testing Therapeutic Toxin Transmembrane Domain Tryptophan United States Food and Drug Administration Variant Vinblastine X-Ray Crystallography absorption biophysical techniques cancer drug resistance cancer therapy clinically relevant combat efflux pump experience experimental study functional group inhibitor insight molecular pump nanodisk novel strategies novel therapeutics prevent protein purification protein reconstitution rational design reconstitution single molecule success summer research tool tryptophan analog undergraduate student uptake

项目摘要

PROJECT SUMMARY/ABSTRACT (of the original funded project) P-glycoprotein (Pgp) is a molecular pump that detoxifies cells by transporting hundreds of structurally unrelated toxins out of the cell. Pgp limits uptake in the intestines, and enhances excretion of drugs in the liver, kidney and blood-brain barrier, of many drugs that are used for treatment of cancers, HIV/AIDS, psychiatric illnesses, and cardiovascular conditions. It is among the seven most important transporters responsible for regulating drug absorption and disposition that now require documentation of drug interactions for approval of any new drugs by the US Food and Drug Administration (FDA). Pgp is an ATP-binding cassette transporter with two transmembrane domains (TMDs) and two nucleotide-binding domains (NBDs). It uses ATP hydrolysis to pump substrates across the cell membranes. Our recent X-ray structures of Pgp identified hydrophobic and aromatic amino acids that contribute to binding of different inhibitors to the drug-binding site. In this proposal, we will test the hypothesis that therapeutic drugs bind to different subsets of residues within defined subpockets in the TMDs of the protein. Our general approach is to introduce tryptophans (Trps) at strategic positions in order to monitor drug binding. The Trps will be introduced on the background of a new fully functional Trp-less Pgp, or a low-Trp Pgp that retains three native conformationally sensitive Trps in the cytoplasmic domains. Using fluorescence changes, such as quenching, and resonance energy transfer (FRET), we will map out sites of interaction of the purified protein with prototypical substrates that occupy biochemically defined and distinct binding sites, as well as those of common therapeutic drugs and newly identified inhibitors. We will further insert a fluorescent Trp analog (L-Anap) into wild-type Pgp using the amber stop codon suppression strategy to explore monitoring drug binding in biological cell membranes. By determining how drugs and inhibitors modulate the cooperativity and conformational dynamics of this multidomain transporter, we will gain unique insight into the mechanisms of drug binding and their effects on Pgp function. With these new approaches, we will address the molecular mechanism and kinetics of drug/inhibitor binding, determine synergistic effects, and refine the mechanisms of drug-drug interactions in Pgp. The information will pave the way to new analytical approaches to refine Pgp drug interaction studies of old and new drugs, and will be invaluable to redesign drugs with clinically favorable pharmacokinetics and accelerate pharmacotherapeutic developments.

项目摘要/摘要（原资助项目）